Use of milk serum apoproteins in the treatment of microbial or viral infection

ABSTRACT

The present invention relates to use of a milk apoprotein or a mixture thereof to prevent or treat microbial or viral infection of the human or animal body. It is believed that this is achieved by inhibiting adhesion of potential pathogens. More preferably, at least one milk apoprotein or a mixture thereof is administered, simultaneously or sequentially, with either or both of at least one free fatty acid or a mixture thereof or a monoglyceride thereof; and/or at least one organic acid or a salt or ester thereof or a mixture thereof. The active agent(s) may be delivered by means of a pharmaceutically acceptable delivery system which includes parenteral solutions, ointments, eye drops, nasal sprays, intravaginal devices, surgical dressings, medical foods or drinks, oral healthcare formulations and medicaments for mucosal applications.

This invention relates to the use of milk apoproteins to prevent ortreat microbial or viral infection of the human or animal body. Morespecifically, these milk apoproteins may be used alone or in combinationwith one or more short chain organic acids and their salts or esterssuch as citric acid and/or one or more free fatty acids and theirmonoesters for inhibiting adhesion and/or growth of potential pathogens.

BACKGROUND ART

Milk is a whitish liquid that is produced from the mammary glands ofmature female mammals after they have given birth. Mammals arewarm-blooded vertebrates of the Class Mammalia, including humans, themammals, for the purposes of the present invention, being morepreferably hoofed, even-toed mammals of the Suborder Ruminantia, such ascattle, sheep, goats, deer and giraffes. Milk from cattle and goats arethe preferred sources of milk apoproteins of the present invention,merely because milk from these sources is readily available on acommercial scale.

Milk serum is a term commonly used in the dairy industry to describe theclear liquid matrix within which casein micelles and butterfat globulesare suspended. Milk serum from ruminants contains the milk sugarlactose; a variety of proteins including milk antibodies, lactoferrinand enzymes; and a variety of lipoproteins including beta-lactoglobulin.Milk serum is a preferred source of milk apoproteins.

Cow's milk is processed in the dairy industry to obtain either butter orcheese. Mechanical agitation is used to break the milk-fat globules toobtain butter, and casein is precipitated to obtain curd from whichcheese is manufactured. The liquid residue remaining after theseprocesses is commonly referred to as milk whey. Milk whey is essentiallythe milk serum, with an increased lipoprotein content arising mainlyfrom the fat globule membrane. Milk whey is a preferred source of milkapoproteins. The term “milk serum apoprotein” as used herein is intendedto embrace milk apoproteins derived from milk serum or milk whey.

There are a variety of different lipoproteins and glycoproteins in milkserum, all of which are characterised by a protein back-bone, to whichlipids and/or carbohydrates are conjugated. Enzymatic hydrolysis may beused to remove the lipids and/or carbohydrates from this proteinback-bone, to prepare the corresponding apoprotein. Although milk serumapoproteins have been isolated, there are no known medical uses for suchmilk serum apoproteins.

Lipids, or fats, include triesters of fatty acids, which may be the sameor different, and glycerol, also described as tri-acylglycerols ortriglycerides. Further hydrolysis may be used to break these esterbonds, thus liberating free fatty acid(s) from the tri-acylglycerols.The use of calf pregastric lipase to liberate free fatty acids from milklipids is reported by Cynthia Q Sun et al (Chemico-BiologicalInteractions 140 (2002), pp185-198). This author reports the growthinhibitory properties of various free fatty acids against Enterococci,which are gram-positive, and coliform bacteria, which are gram-negativebut is silent on the role of milk serum apoproteins.

Free fatty acids are known to exhibit potent antimicrobial and antiviralactivity. In particular, linoleic, linolenic, caprylic and caproic acidswere reported by Schuster et al (Pharmacology and Therapeutics inDentistry 5: pp25-33; 1980) to inhibit the dental caries organism,Streptococcus mutans, and to effect a general reduction in dentalplaque. According to the author, bacteria classified as grain negativeare most sensitive while gram positives are least affected.Additionally, Halldor Thormar et al (Antimicrobial Agents andChemotherapy; January 1987, pp 27-31) review the antiviral properties offree fatty acids and their monoesters, demonstrating the efficacy ofpolyunsaturated long-chain fatty acids and medium-chain saturated fattyacids (and their monoglyceride esters) against enveloped viruses andtheir relative inactivity against nonenveloped viruses, the viricidaleffect being possibly by destabilising the viral envelope itself. Morerecently, the bactericidal activity of free fatty acids was reviewed byR. Corinne Sprong et al (Antimicrobial Agents and Chemotherapy, April2001, pp 1298-1301)—C10:0 and C12:0 fatty acids were found to bepowerful bactericidal agents. The fungicidal properties of C10:0 andC12:0 free fatty acids and their monoglycerides was described byGudmundur Bergsson et al (Antimicrobial Agents and Chemotherapy,November 2001, pp 3209-3212).

Many potentially pathogenic bacteria are common commensals of the skin,hair and mucus membranes—they colonise these areas by adhering to thesurface epithelial cell layer but are normally kept in check by thehost's secretory immune system in mucus and sweat. Disease caused bythese endogenous species usually arises as a result of some debilitationin the host's secretory immune capability, which allows these endogenouspathogens to proliferate.

Adhesion of pathogenic bacteria to host tissue is generally accepted asbeing the first stage in pathogenesis, so that the ability to blockadhesion should be useful in preventing infection. The mechanism of suchadhesion is varied and many organisms employ a multiplicity of bothspecific and non-specific factors. For example, Staphylococci secrete anextracellular teichoic acid, which binds specifically to fibronectin;Candida species employ a glycocalyx of mannoprotein; and Streptococcimake use of water insoluble glucans to colonise the teeth. Because ofthe variety of these factors, it has long been considered impossible todevise a single inhibitor, which would be effective against the widerange of potentially pathogenic species.

The use of antibodies derived by vaccination of some donor animal hasbeen attempted in many situations but, because of the built-inspecificity, the therapeutic use of these antibodies is confined to useagainst the species to which they have been generated.

In all of the above published data, the use of free fatty acids toinhibit growth of a wide range of bacteria, fungi and viruses isdisclosed but there are no known published data disclosing or suggestingtheir efficacy when administered with one or more milk apoproteins inthe inhibition of adhesion and/or growth of potential pathogens in humanand animal healthcare.

The common practice in medical and veterinary care of infection is theapplication of an antibiotic substance designed to inhibit theinfectious agent, which may be fungal, bacterial (both embraced by theterm “microbial”) or viral. In long-term use, many antibiotic substanceshave lost their potency due to the evolution of resistance by theinfectious agent. The problem of antibiotic resistance is most acute inpost-operative situations where the infectious agent is a commoninhabitant of the skin and respiratory tract and, as such, it may havebeen exposed to frequent and varied antibiotics over time, allowing itto evolve resistance to these substances. Large numbers of thesenormally innocuous agents may be disseminated during surgical or nursingprocedures and may give rise to infections when the immune tolerance ofthe patient has been weakened by disease or extended medicalintervention; such infections are frequently described as nosocomialinfections.

One such nosocomial infection is commonly referred to as MRSA(methicillin resistant Staphylococcus aureus). Staphylococcus aureus isa common inhabitant of the respiratory tract of many individuals, whereit is carried asymptomatically without normally causing infection.Because of its ubiquitous nature, it is thought to have been exposed tomany of the commonly used antibiotic substances, and strains now existwhich are resistant to all commonly used antibiotics includingmethicillin. Vancomycin is ‘the drug of last resort’ in MRSA, butstrains have recently emerged that are resistant to vancomycin. Inaddition, vancomycin resistant Enterococcus faecalis (VREF) is a commoninhabitant of the gut and may be disseminated from there during surgicalprocedures, giving rise to other nosocomial infection.

Horizontal gene transfer is a biological term used to describe thepotential transfer of genetic resistance from one species to another.The transfer of antibiotic resistance from species such as VREF topathogenic species such as Clostridium difficile (Pseudomembranouscolitis) is a potentially disastrous event and one which gives cause forgreat concern among the medical profession.

There is therefore a great need for new antimicrobial substances, whichmay be used to treat such antibiotic resistant infections and othersthat are refractive to conventional treatments, and for new antiviralsubstances to treat viral infections for which there are currently feweffective therapeutic remedies.

It is an object of the present invention to retard, preferably block,adhesion of pathogenic organisms and, thus, prevent or treat microbialor viral infection of the human or animal body.

It is a further object of the present invention to combine the retardingor blocking of adhesion with an inhibition of growth, thereby achievingan even greater utility.

It is a still further object of the present invention to achieve theseutilities by the use of a benign material such as, but not limited to,milk serum since this facilitates much more frequent use than isconsidered prudent with many aggressive chemically based medicines.

STATEMENTS OF INVENTION

In a first embodiment, this invention relates to the use of at least onemilk apoprotein to prevent or treat microbial or viral infection of thehuman or animal body. Without wishing to be bound by this, it isbelieved that this is effected by inhibition of adhesion of potentialpathogenic species. Specifically, the milk serum protein back-bone, morecorrectly termed the milk serum apoprotein, which is left after theconjugated lipid and/or carbohydrate is removed from milk lipoproteinsand milk glycoproteins, exhibits potent, broad-spectrum inhibition ofadhesion of potential pathogens to human epithelial cells, as will beexemplified hereunder. When milk serum is used as a source, the residualapoprotein, stripped of its conjugated fatty acids and/or carbohydratemoieties, is amphoteric and inhibits the adhesion of bacteria or otherpathogenic organisms to the host cell surface, thereby preventing firststage pathogenesis. An amphoteric protein has both a hydrophobic(fat-soluble) and hydrophilic (water-soluble) end. The cell surface ofmany microbial species has a lipid or glyco-lipid layer to which thehydrophobic end of the amphoteric protein is attracted. In this way, theapoproteins of milk serum coat the surface of the pathogenic organism,setting up a crude molecular barrier, which prevents the pathogenicorganism (such as a fungus, bacterium or virion) achieving sufficientproximity to the host cell surface to establish adhesion.

Preferably, the aforementioned milk apoproteins are used with free fattyacids or their monoesters (including monoglycerides). As is known, freefatty acids and their monoglycerides are potent antimicrobial andantiviral agents against a wide variety of species, by inhibiting theirgrowth. Thus, simultaneous or sequential (in either order)administration of at least one milk apoprotein and at least one freefatty acid or its monoester inhibits growth, as well as, inhibitsadhesion of a wide range of microbial and viral species. A formulationderived by hydrolysing milk serum or milk whey contains both milkapoprotein(s) and free fatty acid(s).

Alternatively, the aforementioned milk apoproteins are used with shortchain organic acids or their esters or salts. Thus, simultaneous orsequential (in either order) administration of at least one milkapoprotein and at least one short chain organic acid or its ester orsalt inhibits growth, as well as, inhibits adhesion of a wide range ofmicrobial and viral species.

Still more preferably, the aforementioned milk apoproteins may be usedwith both free fatty acids and their monoesters, as well as short chainorganic acids and their salts and esters. Thus, simultaneous orsequential (in any order) administration of at least one milkapoprotein; at least one free fatty acid or its monoester; and at leastone short chain organic acid or its ester or salt inhibits growth, aswell as, inhibits adhesion of a wide range of microbial and viralspecies. A formulation containing all three components can be preparedby hydrolysing milk serum or milk whey.

In this invention, the use of at least one milk serum apoprotein or amixture thereof, optionally with at least one free fatty acid or amonoester thereof or a mixture thereof, and/or optionally at least oneshort chain organic acid or its salt or ester, or a mixture thereof willbe shown to be useful in the treatment of antibiotic resistantinfections of the gastro-intestinal and oropharyngeal tract, the mucosalepithelium and the skin.

While infections from organisms such as MRSA and VREF, and many viralinfections present an acute threat to health, there are seemingly lessinnocuous agents which are common commensals of the body and which maygive rise to discomfort or disease in the longer term. One example ofthis is the generation of dental caries by the bacterium Streptococcusmutans. Dental caries is normally considered a cosmetic problem and istreated as such by the dental profession. There is, however, evidence tosuggest that colonisation of the mouth by Streptococcus mutans maygenerate antibodies which, in systemic circulation, may cross-react withcardiac tissue giving rise to long-term heart disease and autoimmunedamage to other organs.

In this invention the use of milk serum apoprotein(s) are shown toinhibit adhesion of Streptococcus mutans and so provide a useful adjunctin the prophylaxis of dental caries with consequential longer termhealth benefits.

The yeast Candida albicans is a common inhabitant of the skin and mucusmembranes of many individuals where it is carried asymptomatically.Candida colonises the mucus membranes by first adhering to the surfaceof a mucosal epithelial cell from where it proliferates and infiltratesthe cell lumen causing thrush. Constituents of mucus secreted from thesetissues normally inhibit adherence and proliferation; in someindividuals, there is a debilitation of the normal secretory capabilityand a pathogenic process is established. The use of milk serumapoprotein(s) (adhesion inhibitory) and free fatty acid(s) or theirmonoesters and/or organic acid(s) or their salts or esters (both growthinhibitory) will provide suitable prophylaxis in those individualssubject to recurring thrush.

Viral infections do not respond to conventional antibiotics. Whilespecialised anti-viral medicaments are available such as ‘Acyclovir’ forexample used in the treatment of Herpes simplex, in general these areexpensive and limited to a very narrow range of viral infections. Whilemany viral infections have a systemic aspect, some have topical symptomsmanifest as skin rash, blisters and sores, which frequently cause thegreatest discomfort to the individual affected. The use of topicalapplications of formulations containing milk serum apoproteins with freefatty acid(s) and their monoesters will provide local anti-viralactivity which, when used as an adjunct to systemic anti-viraltreatment, will alleviate the external symptoms.

FAMVIR® is a proprietary formulation of Acyclovir (Smith Kline Beecham)designed 20 as a systemic anti-viral agent for oral administration inthe treatment of secondary infections of Varicella zoster (shingles).The Varicella virus causes chickenpox in primary infections withextensive skin eruptions of pus-filled vesicles, which rupture and formscabs. The infection causes intense itching and, when scratched, thesores may leave extensive scaring. The virus remains dormant for manyyears after recovery and may become reactivated by stress orimmunocompromising conditions—the secondary infection is known asshingles and is characterised by an extremely painful skin rash. The useof topical formulations containing milk serum apoprotein(s) and freefatty ester(s) arid monoesters thereof as described herein will minimisethe superficial symptoms on the skin and act as a useful adjunct to theconventional anti-viral therapy. Equally other infections where there isa superficial (skin) dimension such as Rubella (measles) and Herpes(cold sores) arc suitable clinical indications for topical applications.

The Standard Formulation as described hereinafter, which is hydrolysedmilk serum or milk whey, is expected to be effective against pathogenicorganisms at a concentration in the range of 0.5 to 25 mg/ml. It will,of course, be appreciated that the concentration required will depend onthe number of pathogenic organisms to be encountered and the relativeconcentrations thereof. Equally, the concentration ranges desired forthe apoprotein(s); for the free fatty acid(s); and for the organicacid(s) will also depend on the number of pathogenic organisms to beencountered and their relative concentrations.

Free Fatty Acids

A free fatty acid is an organic acid, comprising a hydrocarbon chainwith at least one carboxylic acid functional group, the latter beingusually, although not necessarily, at a terminal position. Fatty acidscan be either saturated, where all carbon to carbon bonds in thehydrocarbon chain are single, or unsaturated, where there is at leastone carbon to carbon double or triple bond in the hydrocarbon chain. Thefree fatty acids or their monoesters are preferably naturally occurringor, alternatively, released by, for example, hydrolysis from naturallyoccurring sources such as, but not limited to, milk serum, egg yolk andvegetable oils.

Preferably, the useful antimicrobial and antiviral free fatty acids aresaturated or unsaturated and have a hydrocarbon chain with an evennumber of carbon atoms (C 4-24), or a mixture thereof.

Suitable unsaturated free fatty acids have a hydrocarbon chain withC14-24 and are preferably selected from palmitoleic (C16:1), oleic(C18:1), linoleic (C18:2), alpha and gamma linolenic (C18:3),arachidonic (C20:4), eicosapentanoic (C20:5) and tetracosenoic (C24:1)acids, in which the bracketed figures represent the number of carbonatoms in the hydrocarbon chain with the number of double (or triple)bonds following the colon representing the degree of unsaturation.

Suitable saturated fatty acids have a hydrocarbon chain with C4-C 18 andare preferably selected from butyric or isobutyric (C4:0), succinic(C4:0), caproic (C6:0), adipic (C6:0), caprylic (C8:0), capric (C10:0),lauric (C12:0), myristic (C14:0), palmitic (C16:0) and stearic (C18:0)acids, which are effective against fungi and the gram-negative bacteria,coliforms and Staphylococci.

It should be appreciated that the free fatty acid(s) or theirmonoesters, whether naturally occurring or not, may be modified bychemical substitution including, but not limited to, short chainalkylation such as methylation or acetylation; esterification; and manyother derivitisations to modify antimicrobial potency and such modifiedfree fatty acids are also intended to form part of the presentinvention. However, for the purposes of the present invention, it ispreferred to use naturally occurring, unmodified free fatty acid(s) ormixtures thereof or their monoesters, preferably their monoglycerides,such as those released from naturally occurring fat reservoirs selectedfrom milk serum, egg yolk and vegetable oils.

Hydrolysis of the lipid content of milk serum provides a suitablemixture of free fatty acids from which broad-spectrum inhibition ofmicrobial and viral growth may be usefully obtained for therapeutic orprophylactic purposes. The following table provides a typical breakdownof the fatty acid composition of milk serum lipid.

TABLE 1 Fatty Acid Composition of Milk Serum Lipid Butyric (C4:0)   4%Caproic (C6:0)  2.1% Caprylic (C8:0)  1.2% Capric (C10:0)  2.6% Lauric(C12:0)  3.0% Myristic (C14:0) 10.6% Palmitic (C16:0)   27% Palmitoleic(C16:1)  2.3% Stearic (C18:0) 12.8% Oleic (C18:1)   26% Linoleic (C18:2) 2.3% Linolenic (C18:3)  1.6% Water Balance to 100%Organic Acids

Suitable organic acids, if present, have a short hydrocarbon chain (forexample, C2-6) with at least one carboxylic acid functional group. Theterm “acid” is intended to embrace their salts or esters. Thehydrocarbon chain may be saturated or unsaturated, straight or branched,substituted or unsubstituted. Suitable organic acids include glycolic,oxalic, lactic, glyceric, tartronic, malic, maleic, fumaric, tartaric,malonic, glutaric, propenoic, cis or trans butenoic, and citric acids.Of the organic acids, citric acid, which is a three-carbon chain withthree carboxylic acid moieties, is preferred. Citric acid is producedduring mammalian metabolism of carbohydrates and is a weak organic acid,which may be neutralised by an alkaline solution such as sodiumhydroxide to give the sodium salt—sodium citrate. As such, it existsnaturally in the body in low concentrations. As shown and claimed inthis invention, when sodium citrate is added to milk serum apoproteinsas described above, the potency of the fatty acids may be amplified withrespect to in vitro cultures of particular bacteria.

Antimicrobial and Antiviral Utilities

Due to the polyspecific nature of inhibition of adhesion which may beachieved using milk serum apoprotein(s), alone or due to thepolyspecific nature of inhibition of adhesion and inhibition of growthusing milk serum apoprotein(s) in combination with free fatty acid(s)and/or organic acid(s), there is a very diverse range of potentialpathogens that may be addressed.

Included among the gram-positive bacteria of significance are theStreptococci, Lactobacilli, Corynebacteria, Propionibacteria,Actinomycetes, Clostridia, Bacillus and Enterococcus.

Gram negatives include Staphylococci and the Enterobacteria,Escherichia, Salmonella, Shigella, and Chlamydia species are alsosensitive.

Among the fungal species, the yeast Candida albicans has been shown tobe sensitive, as well as the dermatophytes including Trichophytonspecies.

The protozoans of significant sensitivity include Entamoeba histolytica,Giardia lamblia and Cryptosporidium neoformatans.

The term “microbial” is intended to embrace bacteria, fingi andprotozoans.

Included among the enveloped virions of significance are Herpes viridae,(Herpes simplex, Varicella-zoster, and Epstein-barr); Poxviridae,(Orthopoxvirus and Avipoxvirus); Togaviridae, (Alphavirus, Flavivirus,Rubivirus and Pestivirus); Coronaviridae, (bronchitis virus);Retroviridae (Human T-cell leukaemia and Human Immunodeficiency virus);Influenza virus, Lyssavirus, California Encaphalitis Virus, Lassa Virus,Paramyxovirus, Pneumovirus and Morbillivirus.

Pharmaceutically and Cosmetically Acceptable Delivery Systems

A pharmaceutically acceptable delivery system comprising apharmaceutically or cosmetically effective amount of at least one milkserum apoprotein, with or without a pharmaceutically or cosmeticallyeffective amount of at least one free fatty acid and their monoestersand/or a pharmaceutically or cosmetically effective amount of at leastone organic acid and their esters or salts may be administered toachieve a clinically useful effect.

Ointments provide a useful delivery mechanism to relieve the superficialsymptoms of viral and bacterial infections manifest in skin rash,blisters and pustules, included among which are herpes, shingles, acneand infectious dermatitis.

Bandages and wound dressings may be impregnated to achieve sustainedrelease of the active material at the site of an infection.

Particularly in the case of methicillin resistant Staphylococcus aureus,the delivery system may comprise a nasal spray for de-contamination ofknown carriers. The use of a delivery system in the form of a skinlotion will provide topical decontamination of skin and hair.

The delivery system may comprise eye drops for the treatment orprevention of infection of the eye.

The delivery system may comprise intravaginal creams or gels, forexample, hydrating and lubricating gels, or pessaries commonly used infeminine care to prevent recurring infections of the yeast Candidaalbicans and as a protection against exogenous bacterial and viraldisease.

The delivery system may comprise a post-surgical wound dressing in whichthe active agent(s) is/are distributed in a sustained release polymer.Such a delivery system may be used to minimise nosocomial infectionsarising from MRSA and other antibiotic resistant bacteria.

The delivery system may additionally comprise antioxidant excipient(s),and be administered parenterally or by IV infusion to achieve a systemicanti-viral and/or anti-microbial effect.

The delivery system may alternatively or additionally comprise amilk-like drink or a food, in which the active agent(s) may be entericcoated to facilitate its transport through the stomach to the intestine,where the active agent(s) can be used as a prophylactic agent againstintestinal infections, including Pseudomembraneous colitis.

The delivery system may comprise oral hygiene products such as chewinggums, mouthwash, toothpaste and denture adhesives and fixatives toachieve reduced caries, and dental plaque and to provide long-termprotection against gingivitis, periodontitis and recurring thrush.

The delivery system may comprise processed foods, in which the activeagent(s) prevent microbial and/or viral spoilage and the potential forfood-borne illness arising from organisms such as Salmonella andCampylobacter.

DESCRIPTION OF THE DRAWINGS

Diagram 1 illustrates four separate size Exclusion chromatograms andthese are marked A, B, C and D.

A). Time 0 hours, pre-treatment: A front running peak at Rt 7.174minutes illuminated at 280 nm has an underlying peak visible at 330 nm.The 330 nm absorbance comes from the lipid moiety conjugated to thelarge proteins that constitute this primary fraction.

B). Time 2 hours after treatment commenced: the primary peak at 7.174minutes has been degraded with contemporaneous increase in two laterunning fractions at Rt 9.7 and 10.6 minutes. The 330 nm lipid fractionhas been degraded and there is no visible increase in the 330 nmabsorbance with the late running peaks.

C). Time 8 hours: shows further degradation of the lipid fraction withno significant change to the late running 280 nm proteins.

D). Time 16 hr: extended incubation shows no change in the overallprofile at either wavelength.

Diagram 2 uses the adhesion of Candida albicans to Buccal EpithelialCell as a measurement of efficacy of the test material, milk serum,before and after enzyme hydrolysis is illustrated. The ‘Control’represents the average total adhesion (36%) achieved with no inhibitorysubstances present. Milk serum before hydrolysis at 1 mg/ml and 2 mg/mlgave some 39% inhibition of the control adhesion (22% adhesion, downfrom 36% adhesion). At 5 mg/ml pre-hydrolysis, 45% inhibition ofadhesion is achieved. The same material is shown at the sameconcentrations after enzymatic hydrolysis. 1 mg/ml is apparently lesseffective than the same material pre-hydrolysis, however at 2 mg/mlthere is 62% inhibition (compared to 39%), and at 5 mg/ml there is 100%inhibition of adhesion, compared to 45% pre-hydrolysis.

Diagram 3 shows the relative inhibition of Candida adhesion achievedfrom each of the lipid and protein fraction of the Standard Formulation.The ‘control’ is at 45% adhesion. The protein fraction at 1 mg/ml isshowing 19% adhesion (58% inhibition), at 2 mg/ml adhesion is down to3%, being 94% inhibition, and totally blocked at 5 mg/ml. In comparison,there is no visible adhesion inhibitory effect from the lipid fractionat any concentration.

Diagram 4 showing, using the same lipid and protein fraction illustratedin Diagram 3, the relative growth inhibitory properties of the lipidfraction on the growth of Candida albicans at 10, 8, and 6 mg/ml. Thereis a progressive increase in growth inhibitory properties asconcentration increases with visible destruction of the yeast culture at10 mg/ml based on loss of Optical Density.

Diagram 5 shows the effect of the protein fraction from Diagram 3 aboveusing the growth of Candida albicans. There is no apparent inhibition ofgrowth arising from the protein fraction at any of the concentrationstested.

Diagram 6 shows the Candida growth inhibitory properties of the StandardFormulation at 0, 1 and 5 mg/ml, 90% inhibition is achieved at thehighest concentration.

Diagram 7 shows an intervention growth assay where the inhibitorysubstances are added after 5 hours of normal growth of Candida albicans.The immediacy of the inhibition at concentrations of 8 and 6 mg/ml isapparent. At lower concentrations the effect is slower but overallinhibition is as effective at 4 mg/ml and some inhibition is alsoevident at 2 mg/ml.

Diagram 8 shows the Candida adhesion inhibitory properties of theStandard Formulation at 1, 2 and 5 mg/ml where the yeast has beenpre-treated by exposure to the test substance for 10 minutes prior tobeing exposed to Buccal Epithelial Cell. At 1 mg/ml there is 53%inhibition of adhesion, while no adhesion occurs at 2 and 5 mg/ml. Theprotein blank in this example is Bovine Serum Albumin at 1 mg/ml showingjust 13% inhibition (relative to the standard formulation at 53%).

Diagram 9 shows the same Standard Formulation used in Diagram 8 above topre-treat the Buccal Epithelial Cells for 10 minutes prior to beingexposed to the Candida culture. At 1 mg/ml there is 55% inhibition ofadhesion while adhesion is totally inhibited at 2 and 5 mg/ml. Theprotein blank in this example is De-ovalbuminised egg white at 1 mg/mlgiving some 12% inhibition of adhesion.

Diagram 10 shows the Standard Formulation inhibiting growth ofmethicillin resistant Staphylococcus aureus (MRSA) by approximately 50%relative to the control where Phosphate Buffered Saline (PBS) is used asa test ‘blank’. When the Standard Formulation is supplemented with 2, 4and 5 mg/ml of sodium citrate, growth is progressively inhibited to zeroat the higher concentrations.

Diagram 11 shows the inhibitory properties of the Standard Formulationagainst adhesion of MRSA to Buccal Epithelial Cell compared with sodiumcitrate and BSA.

At 5 mg/ml the Standard Formulation achieves 98% inhibition of adhesion,while at the same concentration sodium citrate is inhibiting atapproximately 10%, there is no effect from the protein blank.

Diagram 12 shows that the growth of the dental caries causing organismStreptococcus mutans is inhibited by 5 mg/ml of the StandardFormulation, under the test conditions described in the text.

Diagram 13 shows that Streptococcus mutans adheres to hydroxyapatitebeads, used here as a surrogate for dental enamel. The standardFormulation at 0.8 mg/ml achieves approximately 100% inhibition ofadhesion under the test conditions. Sodium citrate affects adhesion ofthis organism by some 10% at 0.8 mg/ml while Bovine Serum Albumin usedas a protein blank achieves some 30% inhibition under these testconditions.

Methods and Materials:

Milk serum proteins may be extracted from whole fresh milk, preferablyfrom whole fresh ruminant milk, by first separating the butter-fat usingcentrifugation. The supernatant is then acidified to pH 4.5, at whichpoint the caseins precipitate. Further centrifugation will leave a clearsupernatant containing the milk sugar lactose, the milk serum proteinsand dissolved minerals. Lactose, which represents a substantialproportion of the solids content of milk serum (up to 50%), is thenremoved by dialysis or ultra-filtration. The resultant “conjugatedprotein-rich” fraction will have a composition approximating to thefollowing (v/v):

Beta-Lactoglobulin 56% Alpha-lactalbumin 11% Gamma-globulin 12% Serumalbumin  6% Lactoferrin  4% Mucins  2% Enzymes  1% Minor proteins  1%Protein bound lipid (fat)  7%

Many of these proteins are complex lipoproteins or glycoproteins withsubstantial non-protein macromolecules conjugated to them, but the majorprotein component of whey from ruminant mammals is beta-lactoglobulin,which may represent up to 70% of whey and 90% of colostrum (the firstmilk after parturition). Beta-lactoglobulin is a lipoprotein, withsubstantial amounts of the isoprenoid, retinol, conjugated to it, butlipids and fatty acids make up a substantial portion of the non-proteincomponent.

Alternatively, a convenient source of milk serum proteins is dairyindustry whey powder, which may be obtained commercially from manydifferent sources. In many cases, commercial suppliers have alreadyremoved the lactose content, providing a “conjugated protein-rich”material with a fat content of between 6 and 10%, such material beingthe preferred source material for use in this invention. Whilst somecommercial suppliers use Ultra High Temperature (UHT) to increase shelflife of liquid whey, such treatment denatures the protein back-bone andrenders the material useless for the purposes described herein. Wherethe fat content is below 6%, it may be supplemented by adding butterfatback to the whey powder, when it is re-constituted (in purified water(water purified by reverse osmosis)) for enzyme hydrolysis as describedherein.

A suitable commercial source of standardised low lactose whey powder is‘Carbelac 80’ a whey protein concentrate from Carbery Milk Products,Ballineen, County Cork, Ireland.

Inhibition of Growth Assay

The inhibition of growth of various bacteria or yeast may bedemonstrated by growing the organism in a suitable medium with andwithout test substances such as milk serum apoproteins, free fattyacids/monoesters and/or organic acids/salts/esters; the test formatbeing suitably constructed with media blanks and controls. A microtitreplate assay may be used to increase the number of test points and growthis measured using optical density determination.

In order to ensure that there is no dilution effect arising from theaddition of different concentrations of test material, test solutionsare prepared by dissolving or suspending the appropriate amount of testsubstance, in the appropriate fresh growth medium for that bacteria oryeast. Typically, test solutions are prepared from a stock solution witha concentration of 20 mg/ml, prepared by dissolving, for example, 200 mgof test substance in a final volume of 10 ml of fresh growth medium. Thestock solution is centrifuged at 6,000 rpm for 10 minutes to removesuspended solids. The stock solution is then aseptically diluted usingappropriate volumes of the stock solution and fresh growth medium, toachieve test concentrations of, for example, 10, 8, 6, 4, 2 mg/ml.

The dilution step to achieve say 10 mg/ml from a 20 mg/ml stock solutionis described herein as 1:2 and by this is meant one volume of stocksolution is diluted with 1 volume of diluent, to achieve a final volumeof 2 volumes. This convention will be used herein to refer to alldilution steps used herein.

Prepared solutions are pre-warmed to 37° C. 100 microliters is added toeach well as required immediately before addition of 100 microliters ofthe prepared inoculum. Thus, a test concentration of 10 mg/ml issubjected to a further 1:2 dilution in the test well, so that the “10mg/ml” is actually 5 mg/ml in the test well itself. The Multiskan Ascenthas an automatic shake cycle that is used to ensure even distribution ofculture prior to each OD reading.

Concerning inhibition of growth of Candida albicans, a ‘Nunc’ 96 wellmicrotitre plate (Nalge Nunc International, Copenhagen, Denmark) isused, each well of which holds the aforementioned 200 microliters. Testpoints are conducted in quadruplicate. The inoculum consists of 100microliters of freshly grown bacteria, or yeast prepared as describedbelow. The final volume in each well consists of a total of 200microliters, comprising 100 microliters of the appropriate dilution oftest substance and 100 microliters of inoculum in fresh medium.

Inoculated plates are loaded into a ‘Multiskan Ascent’ (LabSystems,Helsinki, Finland) incubated microtitre plate reader and held at 37° C.for a period of up to 18 hours during which optical density changes inthe wells are measured at 600 run every hour. At the end of the growthcycle, the results are processed by averaging each of the quadruplicatewells, and illustrating the changes graphically.

Yeast:

12 hour (overnight) culture of Candida albicans in Oxoid Yeast MinimalMedia with 5% (w/v) glucose added (Oxoid is a trademark), diluted 1:10(one to a final volume of 10) (v/v) with fresh medium at 37° C., add 100microliters to each well.

The yeast Candida albicans has an optimal pH for growth of between 4.0and 4.5 and many of its pathogenic processes are also optimal in this pHrange. The growth assay described above may be modified with the use of50 mM sodium lactate buffer at pH 4.0 to prepare the yeast minimal mediaand the test solution, so as to more adequately reflect the in vitroenvironment wherein the Standard Formulation will be effective.

A similar methodology may be used to measure growth, and inhibition ofgrowth, of bacteria (and fungi) by modifying the growth mediaaccordingly. In the case of Streptococcus mutans and Staphylococcusaureus, both shown herein as examples, the growth media is Oxoid BrainHeart Infusion Broth (Oxoid is a trademark).

Adhesion Assay:

Measurement of adhesion, and inhibition of the same, requires selectionof a suitable substrate and a method of enumerating number of organismsadhering (or not) to that substrate. Most potential pathogenic organismsadhere to mucosal epithelial cells and a convenient source of arepresentative mucosal epithelial cell may be easily harvested frominside the cheek. Buccal epithelial cells (BEC's) are harvested byscraping the mucosal membranes in the mouth using a wooden tonguedepresser as follows.

Harvesting of Buccal Epithelial Cells:

Standard wooden tongue depressers as used in clinical examination of themouth are wrapped in tin foil and autoclaved. 5.0 ml aliquots of 0.1Mpotassium phosphate containing 0.9% (w/v) sodium chloride pH adjusted to6.8 (i.e. PBS) are placed in sterile 25 ml sample bottles. Tonguedepressers are then used to rub the inside of a volunteer's cheek andthe collected scrapings are transferred to the PBS containers. Thecollected samples are centrifuged at 1,000 rpm for 3 minutes to sedimentthe BEC's, leaving bacteria and other oral detritus in suspension. Thesupernatant from these tubes is decanted and a further 5 ml of freshsterile PBS added, the BEC's are re-suspended and re-centrifuged twiceto achieve ‘washed cells’.

There are many different methods of enumerating bacteria and yeast, allof which are well known to those skilled in the art and these includeviable plate counting, direct microscopic counting, radio-scintillationlabelling and fluorescent labelling. Any validated method of enumerationis suitable, provided it does not interfere with the organism's abilityto adhere to the chosen substrate.

In the examples given hereunder, direct microscopic counting has beenchosen for the yeast Candida and a fluorescent label for bacteria. Moreimportantly, however, the number of yeast and bacteria not adhering froma standard population have been enumerated, as distinct fromendeavouring to count adhering cells, because the substrate usuallyinterferes with the count.

The basis of the technique involves exposing a standardised (known)number of yeast or bacteria to a standardised substrate (number ofBEC's), allowing a 60 minute incubation period for the cells to adhereand then filtering the combined population through a 10 micron nylonmesh. The mesh will retain BEC's and those yeast or bacteria adhering tothem, non-adhering yeast or bacteria will be washed through, where theymay be enumerated in the filtrate and expressed as a percentage of theoriginal population not adhering; percent adherence being the inverse ofthis.

Direct microscopic counting of yeast and BEC is conducted using agraduated haemocytometer slide and the method is well known to thoseskilled in the art.

Fluorescent labelling of bacteria is conducted after adhesion (on thosecells in the filtrate) using fluorescent dyes such as BCECF/AM(Calbiochem Biosciences Inc., La Jolla, Calif.) and Syto 13 (MolecularProbes, Oregon, USA). Labelling methods are as described by themanufacturers of the dyes. The amount of fluorescence is measured in afluorimeter (Fluroscan from Lab-Systems, Helsinki, Finland) and is indirect relationship to the number of bacteria present.

The following general method may be used to measure adherence of theyeast Candida albicans to BEC and the inhibition of that adherence usingthe formulation of milk serum apoprotein(s) as described above. The samegeneral method is suitable for measuring inhibition of adherence ofStaphylococcus aureus to BEC's and also inhibition of adherence ofStreptococcus mutans, with the exception that the substrate forStreptococcus mutans, is powdered hydroxylapatite available from Merckand used as a surrogate for dental enamel.

A fresh clinical isolate of Candida albicans is preferred as many of thetype cultures have lost virulence in culture collections. If a clinicalisolate is not available, C. albicans type strain ATCC 10231 may be usedto achieve representative results (ATCC is the American Type CultureCollection housed in Maryland USA).

The yeast is routinely cultured on Oxoid Malt Extract Agar (Oxoid is atrademark). Oxoid Yeast Extract Peptone Dextrose broth is used forliquid cultures, these are inoculated from fresh agar plates andincubated with agitation at 37° C. for 10 hours. After ten hours, theyeast is harvested by centrifugation and washed in sterile PBS at pH6.8.

Both the washed BEC's and the freshly grown washed yeast cells arecounted microscopically and the concentrations adjusted such that theyeast is in the order of 1×10⁵ and BEC's at 1×10³. Equal volumes of thetwo solutions when mixed will give a ratio of 100 yeast cells per BEC.

In testing various concentrations of test substances such as milk serumapoprotein(s), these are added in the desired strength to the PBS (pH6.8) used in the final suspension of either yeast or buccal cells.Typically, concentrations of 5, 2 and 1 mg per ml are used and tests areconducted to determine the effectiveness of the formulation atinhibiting adhesion by pre-coating BEC's or pre-coating the yeast. Aperiod of just ten minutes is allowed as ‘pre-coating’ prior tocombining with the other of the two suspensions when adhesion begins.The formulation is shown to be effective in either pre-coating of BuccalEpithelial Cells or pre-coating of Candida albicans. Thus, greaterutility may be achieved by creating a protective molecular barrier oneither or both of these surfaces. Specifically, pre-coating BuccalEpithelial Cells will prevent adhesion being established whilstpre-coating the pathogenic organism, in this case yeast, will prevent analready established colony from extending to other areas.

Equal volumes of the two solutions/suspensions are mixed and incubatedwith gentle agitation for 60 minutes, after which the mixture isfiltered through a nylon mesh with a defined porosity of 10 microns. Thenumber of yeast in the filtrate are counted microscopically andexpressed as a percentage of the original population. When highvirulence clinical isolates are used, it is not unusual to achieve up to40% adherence.

EXAMPLE 1 Preparation of Milk Serum Free Fatty Acids and Milk SerumApoproteins

Lactose free whey powder (Carbelac 80 from Carbery Milk Products) is thestarting material. Carbelac 80 is typically 100% whey, of whichtypically 0% is skim, 80% is protein, 5% is moisture, 8% is fat and 3%is ash. 30 grams of this starting material is dissolved to a finalvolume of 1 liter of phosphate buffered saline (PBS) at pH 6.8. To thisis added 1 gram of a suitable composition of various esterase (mainlylipases, but also amylases and proteases) enzymes, for example, ‘LipaseType 2 Crude from porcine pancreas’ available from Sigma. The mixture isincubated at 37° C. for 18 hours; heat-treated at 60° C. for 10 minutesto inactivate the enzyme; and spray dried.

Other suitable esterase enzymes include, but are not limited to,‘Palatase’ and ‘Novozyme’ commercially available from Novo Nordisk,Copenhagen, Denmark and used in a 50:50 mix (w/w) at 1 gram per 30 gramof lactose free whey.

The course of the apoprotein and free fatty acid/monoglyceride formationmay be followed chromatographically as illustrated in Diagram 1. Gelfiltration (size exclusion) HPLC using Sephacryl S-200 and PBS (pH 6.8)as an elution buffer will give adequate resolution to illustrate themain events during enzymatic hydrolysis. Use of two wavelengths tomonitor the eluent is advantageous; at 280 nm the proteins areilluminated while use of a 330 nm wavelength illuminates thelipid/carbohydrate component conjugated to these proteins. A frontrunning peak at Rt (retention time) 7.174 minutes (Diagram 1 A)represents the early elution of large proteins and the lipid componentis visible as an underlying peak. During the course of hydrolysis(Diagrams 1B (2 hours) and 1C (8 hours)), the front peak and itsconjugated lipid/carbohydrate disappear, with a commensurate increase inthe concentration of two late running fractions (280 nm) at 9.7 and 10.6minutes, these being smaller proteins and the apoproteins from the frontpeak. Diagram 1D shows that extended hydrolysis (16 hours) shows nofurther change in Rt at either wavelength.

Using procedures as outlined above, a typical composition of enzymetreated lactose free whey (or apoprotein-rich and free fatty acid-richfraction) will consist of the following “Typical Formulation”.

COMPONENT % (v/v) Apoprotein of Beta-lactoglobulin 25-35 Apoprotein ofFat Globules  5-15 Free fatty acids 15-25 (see Table 1) Residual Lipid(includes cholesterol)  5-15 Apoprotein of Alpha-lactoglobulin  5-15Apoprotein of Gammaglobulin  6-10 Serum albumin  1-3 Alpha-tocopherol 2-6 Sodium citrate  2-6 Sodium phosphate  1-3

The apoprotein preparation procedure may be enhanced by the addition ofsurfactants such as purified components of bile salts such as cholicacid and/or by the addition of suitable enzyme co-factors such ascalcium salts and/or by the addition of suitable buffers such as sodiumcitrate. In some applications such as the inhibition of growth of MRSA,the residual sodium citrate also contributes to growth inhibitoryproperties but not inhibition of adhesion. The stability of the freefatty acids and their monoglycerides may be enhanced by the addition ofanti-oxidants such as, for example, alpha tocopherol (vitamin E).

The gamma-globulin (immunoglobulin) content of whey may be manipulatedby immunisation of the donor animal. Immunisation procedures are wellknown and the specificity of the immunoglobulins may be tailored andamplified towards any particular organism using attenuated strains ofthat organism in the vaccine. Whilst the use of such immune wheys fallwithin the scope of the present invention, the use of non-immune whey,where the ‘native’ gamma-globulin has no particular specificity for anyorganism, is preferred.

This resulting hydrolysed material exhibits inhibition of growth andinhibition of adhesion as illustrated in the following Examples usingthe Streptococcus mutans, the dental caries organism, the yeast Candidaalbicans and methicillin resistant Staphylococcus aureus.

The “Standard formulation” as described and exemplified hereinaftercomprises (v/v):

Apoprotein of Beta-lactoglobulin 32% Apoprotein of Fat Globule Membrane 8% Free fatty acids 22% (see Table 1) Residual Lipid (includescholesterol)  8% Apoprotein of Alpha-lactoglobulin 10% Apoprotein ofGammaglobulin  8% Serum albumin  2% Alpha-tocopherol  5% Sodium citrate 3% Sodium phosphate  2%

The hydrolysis procedure activates adhesion inhibitory properties thatare not present in pre-activated whey. Diagram 2 illustrates thiseffect, by comparing pre-hydrolysis and post hydrolysis milk serum (thelatter being Standard Formulation) on the inhibition adhesion of Candidaalbicans to Buccal Epithelial Cells. As will be observed, whilstunhydrolysed milk serum shows some inhibition of adhesion, there is amarked, concentration-dependent inhibition of adhesion in the presenceof hydrolysed milk serum, so that no adherence of Candida albicans isdetected at 5 mg/ml.

Separation of Milk Serum Apoproteins:

The Standard Formulation may be fractionated using a chloroform:methanol extraction procedure to separate the lipid and apoproteinfractions.

The procedure was performed using a concentration of StandardFormulation at 10 mg/ml of phosphate buffered saline at pH 6.8. One mlof this solution was added to glass tubes containing 5 ml of chloroformand 2.5 ml of methanol. The mixture was vortexed for 30 seconds and thenagitated for 30 min's, after which it was allowed to stand untilseparation of the solvent layers was complete. Using a Pasteur pipette,the upper methanol layer was removed. Aliquots of each solvent fractionwere vacuum dried. Any polar compounds (proteins) are present in thepolar solvent (methanol) fraction, and non-polar (fatty acids) will beretained in the chloroform layer. The dried methanol fractions weretaken up in one tenth their original volume in phosphate buffered salineat pH 6.8 and the dried chloroform fraction was taken up in one tenththe original volume of ethanol, and diluted in PBS for test purposes.

Diagram 3 illustrates the adhesion inhibitory properties of both theapoprotein-rich and lipid-rich fractions, concentration-dependentinhibition of adhesion being associated with the apoprotein-richfraction and not the lipid fraction. Indeed, at 5 mg/ml, virtually noadhesion of Candida albicans could be detected.

Diagram 4 illustrates the concentration-dependent growth inhibitoryproperties of the lipid-rich fraction on Candida albicans, while Diagram5 shows that the apoprotein-rich fraction has no effect on growth (thereis in fact some amplification of growth at 10 mg/ml from the increasingconcentration of protein fraction).

EXAMPLE 2

Using the growth assay described in Methods and Materials above, thegrowth inhibitory properties of the Standard Formulation against a freshclinical isolate of Candida albicans was evaluated. The assay was amicrotitre plate format and each test concentration was conducted inquadruplicate. Growth was measured at 600 nm over a 20-hour period andthe results are illustrated in Diagram 6. The Standard Formulation (at 5mg/ml) gave almost 90% inhibition of growth, relative to the controlwith 0 mg/ml Standard Formulation added. The Standard Formulation at 1mg/ml gave an intermediate result.

Using a similar assay procedure, with the exception of adding the testsubstances after 5 hours of normal growth, is described herein as anintervention assay. The Standard Formulation is added at concentrationsranging from 0 mg/ml to 8 mg/ml. The test concentrations are set-up suchthat there is similar dilution effect in all wells when pre-warmed (to37° C.) test solutions are added. The results of an intervention assayon a fresh clinical isolate of Candida albicans are presented in Diagram7, the data having been processed to remove the optical density changeat 5 hours resulting from the addition of test material. The immediateand dramatic, concentration dependent, inhibitory effect of the StandardFormulation on the growth of C. albicans is evident at concentrations of8, 6, 4 and 2 mg/ml.

EXAMPLE 3

The Standard Formulation inhibits adhesion as well as growth. Theadhesion assay method has been described in Methods and Materials above.Using the same formulation as in Example 2, the inhibitory effects onthe adhesion of the same fresh clinical isolate to BEC's are illustratedin Diagrams 8 and 9.

In Diagram 8, the yeast cells have been exposed to the StandardFormulation for 10 minutes prior to being added to the BEC's. In Diagram9, BEC's have been exposed to the Standard Formulation for 10 minutesprior to being added to the yeast suspension.

The ‘Control’ in both of these assays represents the adhesion achievedunder the test conditions when no inhibitory substances are present; 41%and 35%, respectively. The addition of Standard Formulation at 1 mg/mlin Candida pre-treatment reduces adhesion to 20% (53% inhibition), whilethe same concentration in BEC pre-treatment reduces adhesion to 16% (55%inhibition). At 2 and 5 mg/ml of Standard Formulation in bothpre-treatment of yeast and BEC's, there is 100% inhibition of adhesionunder the test conditions.

The “protein blank” in Diagram 8 is Bovine Serum Albumin and in Diagram9, de-ovalbuminised egg white was used, both at 1 mg/ml, and bothintended to indicate that the effect of the Standard Formulation is not‘simply’ an effect due to protein concentration.

EXAMPLE 4

The effectiveness of the Standard Formulation against both growth andadhesion of methicillin resistant Staphylococcus aureus (MRSA) isdemonstrated in Diagrams 10 and 11.

MRSA is routinely sub-cultured on blood agar and a single colony is usedto inoculate a tube of Oxoid Brain Heart Infusion Broth as described inMethods and Materials above. After 8 hours, the innoculum is used ingrowth and adhesion assays using the methodologies described above.

MRSA is not as sensitive to free fatty acids/monoglycerides as otherorganisms and the addition of citrate salts, as are contained in theStandard Formulation, are essential for meaningful inhibition of growthof this particular organism.

Diagram 10 illustrates the effect of the Standard Formulation at 5 mg/mland with increasing concentrations of trisodium citrate (0, 2, 4 and 5mg/ml), complete inhibition of growth is achieved when trisodium citrateat 4 or 5 mg/ml is added to Standard Formulation of 5 mg/ml. Trisodiumcitrate is added to the test solutions while they are being prepared inthe growth medium as described under “growth assay” methods.Specifically, as described, the stock solution of Standard Formulation(concentration of 20 mg/ml) is mixed with an equal volume of a trisodiumcitrate solution (concentration 20 mg/ml). Thus, a 1:2 dilution isachieved in a solution containing 10 mg/ml of Standard Formulation and10 mg/ml trisodium citrate. As explained above, this compositeformulation is, in the test well, a 5 mg/ml Standard Formulation and a 5mg/ml trisodium citrate. Equally, of course, the stock StandardFormulation solution of 20 mg/ml may be mixed with an equal volume of atrisodium citrate solution containing 16 mg/ml or 8 mg/ml, so as toachieve a Standard Formulation supplemented with 4 and 2, respectively,mg/ml trisodium citrate as is shown in Diagram 10.

MRSA adheres to BEC's, and these are used here in a manner similar tothat described for Candida albicans. Sodium citrate, alone, has noeffect on adhesion of MRSA to BEC whilst, as illustrated in Diagram 11,almost complete inhibition of adhesion is achieved at 5 mg/ml of theStandard Formulation (no added sodium citrate) under the testconditions. Bovine Serum Albumin is used as a ‘protein blank’ and thismaterial has no effect on the adhesion of MRSA to BEC's.

EXAMPLE 5

The organism Streptococcus mutans is considered to be the causativeagent of dental caries, since it adheres avidly to the enamel surface ofteeth. Fermentation of carbohydrate results in the secretion of lacticacid, bringing localised pH down to where there is dissolution of thedental enamel and the onset of dental caries.

Diagram 12 illustrates the effect of 5 mg/ml of the Standard Formulationon the growth of Streptococcus mutans. The test is conducted in themanner described for all growth assays above. At 5 mg/ml of StandardFormulation, there is complete inhibition of growth of the organism overthe 15 hours assayed. During the same period, the control growth, withPhosphate Buffered Saline at pH 6.8 added instead of the StandardFormulation, shows the expected logarithmic increase.

In measuring inhibition of adherence of Streptococcus mutans,hydroxyapatite powder from Merck was used as a surrogate for dentalenamel. The test procedure is as described in Methods and Materialsabove, with the following modifications: 1 ml of fresh culture, adjustedto an optical density of 0.1 at 600 nm, is added to 5 mg of salivacoated hydroxyapatite beads; allowed to adhere for 1 hour; andcentrifuged at slow speed to sediment the hydroxyapatite with adheringbacteria. The number of bacteria remaining in the supernatant are thatpercentage of the original population not adhering and are expressed asa percentage of the original population; the inverse being the percentinhibition of adherence.

Diagram 13 illustrates the dose-dependent, adhesion inhibitory effect ofthe Standard Formulation; almost complete inhibition being achieved at0.8 mg/ml. Again in this example, Bovine Serum Albumin was used as a‘protein blank’ and, in this test system, BSA is having an inhibitoryeffect of some 30% at 0.8 mg/ml, whereas sodium citrate shows some 10%inhibition of adhesion.

1. A pharmaceutically acceptable delivery system comprising: at leastone milk serum apoprotein selected from the group consisting of a)apolipoproteins, b) apoglycoproteins, and c) a mixture of a) and b),whereby said milk serum apolipoprotein is the protein moiety remainingafter conjugated lipid is removed from milk serum lipoproteins, and saidmilk serum apoglycoprotein is the protein moiety remaining afterconjugated carbohydrate is removed from milk serum glycoproteins; and apharmaceutically acceptable carrier.
 2. The delivery system according toclaim 1, in which said milk serum apoprotein is an apolipoprotein. 3.The delivery system according to claim 2, in which said apolipoproteinis derived from a milk serum lipoprotein selected from the groupconsisting of beta-lactoglobulin, fat globule membrane, and a mixturethereof.
 4. The delivery system according to claim 1, in which said milkserum apoprotein is prepared by hydrolyzing milk serum or milk whey withan enzyme(s); denaturing the enzyme(s); and separating theapoproteins(s)-rich fraction.
 5. The delivery system according to claim4, in which the enzyme is a lipase.
 6. The delivery system according toclaim 1, in which said milk serum apoprotein is prepared by hydrolyzingmilk serum or milk whey with an enzyme(s).
 7. The delivery systemaccording to claim 6, in which the enzyme is a lipase.
 8. The deliverysystem according to claim 1, in which said milk serum apoprotein is fromcow or goat milk.
 9. The delivery system according to claim 1, furthercomprising at least one additional component selected from the groupconsisting of a free fatty acid and a monoglyceride thereof.
 10. Thedelivery system according to claim 9, in which said free fatty acid orthe monoglyceride thereof is either saturated or unsaturated and has ahydrocarbon chain with an even number of carbon atoms numbering between4 to 24 carbon atoms.
 11. The delivery system according to claim 10, inwhich said free fatty acid or the monoglyceride thereof is anunsaturated fatty acid having a hydrocarbon chain with a number ofcarbon atoms numbering between 14 and 24 carbon atoms.
 12. The deliverysystem according to claim 9, in which said free fatty acid or themonoglyceride thereof is selected from the group consisting ofpalmitoleic, oleic, linoleic, alpha and gamma linolenic, arachidonic,eicosapentanoic, and tetracosenoic acids, and their monoglycerides. 13.The delivery system according to claim 10, in which said free fatty acidor the monoglyceride thereof is a saturated fatty acid having a carbonchain with a number of carbon atoms numbering between 4 and 18 carbonatoms.
 14. The delivery system according to claim 9, in which said freefatty acid or the monoglyceride thereof is selected from the groupconsisting of butyric, isobutyric, succinic, caproic, adipic, caprylic,capric, lauric, myristic, palmitic, and stearic acids, and theirmonoglycerides.
 15. The delivery system according to claim 1, furthercomprising at least one additional component selected from the groupconsisting of an organic acid, an organic acid salt, and an organic acidester.
 16. The delivery system according to claim 15, in which saidorganic acid, organic acid salt, or organic acid ester is selected fromthe group consisting of glycolic, oxalic, lactic, glyceric, tartronic,malic, maleic, fumaric, tartaric, malonic, glutaric, propenoic,cis-butenoic, trans-butenoic, and citric acids, and their correspondingsalts and esters.
 17. The delivery system according to claim 1, furtherincorporating an anti-oxidant.
 18. The delivery system according toclaim 17, in which the anti-oxidant is alpha-tocopherol.
 19. Thedelivery system according to claim 6, in which the hydrolyzed milk serumor milk whey is present in a concentration range of 0.5 to 25 mg/ml. 20.The delivery system according to claim 1, in which the milk serumapoprotein is present in a concentration range of 0.5-10 mg/ml.
 21. Thedelivery system according to claim 20, in which the milk serumapoprotein is present in a concentration range of 3-7 mg/ml.
 22. Thedelivery system according to claim 9, in which said free fatty acid orthe monoglyceride thereof is present in a concentration range of 0.5-5mg/ml.
 23. The delivery system according to claim 15, in which saidorganic acid, organic acid salt, or organic acid ester is present in aconcentration range of 0.5-5 mg/ml.
 24. The delivery system according toclaim 9, in which said milk serum apoprotein and said additionalcomponent, selected from the group consisting of a free fatty acid andthe monoglyceride thereof, are administered either simultaneously orsequentially within 6 hours in either order.
 25. The delivery systemaccording to claim 15, which system has at least two components, inwhich said milk serum apoprotein is one component and said additionalcomponent, selected from the group consisting of an organic acid, anorganic acid salt and an organic acid ester, as the second component,are administered either simultaneously or sequentially within 6 hours ineither order.
 26. The delivery system according to claim 9, which systemhas at least two components, further incorporating at least oneadditional component, selected from the group consisting of an organicacid, an organic acid salt, and an organic acid ester, to form a systemcomprising at least three components; and in which said milk serumapoprotein, as the first component; said free fatty acid or themonoglyceride thereof, as the second component; and said organic acid,organic acid salt, or organic acid ester, as the third component; areadministered either simultaneously or sequentially within 6 hours ofeach other in any order.
 27. The delivery system according to claim 26,in which said first component comprises apolipoproteins; said secondcomponent comprises free fatty acids; and said third component comprisesan organic acid that is citric acid or its salt.
 28. The delivery systemaccording to claim 27, in which said first, second and third componentsof said delivery system are administered simultaneously.